Chronic exposure to nicotine leads, in some individuals, to the set of behaviors that constitute nicotine dependence. Chronic nicotine exposure produces selective upregulation of certain high-sensitivity (HS) nicotinic acetylcholine receptors (nAChRs). This project works within an emerging hypothesis that selective upregulation of certain nAChRs is both necessary and sufficient for the initial stages of nicotine dependence- minutes, hours, days, and weeks. The power of the emerging hypothesis derives from the selectivity displayed by upregulation at every level thus far examined-brain region, cell type, subcellular region, major pharmacological subtype, and detailed subunit composition. These are tiers of selectivity in upregulation. As a result, upregulation selectively modifies neuronal excitability and neuronal interactions. These pathological patterns comprise, in part, the cellular and circuit basis of nicotine dependence. Aim 1 studies selective upregulation in the ventral tegmental area-nucleus accumbens (VTA-NAcc) pathway, which accounts for some aspects of both tolerance and sensitization to the rewarding aspects of nicotine. Previous data, especially on the substantia nigra-dorsal striatum pathway, show tiers of selectivity in upregulation. Now five linked sub- hypotheses will be tested about the cellular and subcellular details of alpha4beta2 nAChR upregulation in the VTA and NAcc. Aim 2 tests hypotheses about upregulation in medial entorhinal cortex and dorsal hippocampus, a partial model for cognitive sensitization during chronic exposure to nicotine. Sub-hypothesis 2.1 further characterizes the known upregulation of a4* nAChRs by chronic nicotine on the axons of the medial perforant path. This upregulation leads to increased glutamate release in the presence of nicotine. Sub- hypothesis 2.2 states that chronic nicotine upregulates a4* nAChRs in some hippocampal interneurons. Aim 2 also characterizes upregulation in dorsal hippocampus after only 24 hours of nicotine exposure. Aim 3 tests hypotheses about the amygdala, which is a partial locus for nicotine's calming responses to environmental stress. The project tests whether nicotine effects in amygdala arise partially via alpha4beta2 nAChRs on axon terminals of dopaminergic VTA projections. Experiments will focus on two amygdala cell types with dense dopaminergic innervation: intercalated neurons, and corticotrophin-releasing factor neurons in central amygdala, lateral region. The project then tests whether upregulation of these nAChRs amplifies the response to acute nicotine, providing a form of sensitization to the calming effects of nicotine during environmental stress. Aim 4 tests a hypothesis linking the alpha5 subunit to aspects of nicotine addiction. Specifically, the project tests whether lower levels of the alpha5 subunit produce more alpha4beta2 nAChRs, and therefore greater upregulation by chronic nicotine. The techniques, all established in the investigator's laboratory, will be applied as appropriate: a. Patch-clamp electrophysiology in brain slices; b, Electrochemical measurements of DA release; c, Quantitative fluorescence measurements in existing mouse strains with fully functional a4* receptors; d, Measurements in KO mice for the a5 subunits, and selectively in other strains; e, Multielectrode array-based single-unit extracellular recordings from intact mice. This project is a part of a broad context. Tobacco use presents challenges requiring data at many levels-from genome-wide association studies, through the intracellular molecular bases of upregulation, through more complex circuits, through animal and human behavior, through public policy. PUBLIC HEALTH RELEVANCE: Roughly 400,000 Americans and roughly 4,000,000 people worldwide die annually of the sequelae to nicotine dependence. This project attempts to explain nicotine dependence.